Structural Control of Dual Emission in Coumarin Fluorophores for Visualizing Protein Droplet Maturation

Proteins droplets formed by liquid-liquid phase separation (LLPS) gradually lose fluidity and mature into aggregated and fibrillar states, initiating fibrillation of amyloidogenic proteins. Despite increasing interest in droplet maturation, high-throughput methods for visualizing this process remain undeveloped. In this study, we report a fluorescence-based method for real time visualization of droplet maturation using dual-emission fluorophores. Coumarin-based fluorophores conjugated with electron-withdrawing rings exhibit dual fluorescence originating from multiple ground-state conformers although predicting this behavior from molecular structure remains challenging. Here, we synthesized coumarin-based fluorophores bearing directly conjugated electron-withdrawing five-membered ring to increase the abundance of twisted conformers through steric hindrance. The dual-emission properties and fluorescence quantum yield were tuned by varying the heteroatoms within the five-membered ring. Upon covalent conjugation of the probe to proteins, ratiometric confocal microscopy revealed that the emission ratio faithfully reflected the maturation process of liquid droplets, enabling quantitative evaluation of maturation timescale and heterogeneous internal environments. Furthermore, by combining this probe with the photoactive yellow protein (PYP) tag strategy, we visualized differences in the internal environments of protein droplets in living cells. This strategy allows continuous monitoring of droplet maturation and provides new insight into the mechanism of LLPS, amyloidogenic proteins fibrillations, and inhibitor screening against amyloid formation.